The oligosaccharides (glycans) conjugated to proteins and cellular wall components play important roles in cellular signaling and biological activity. For example, the detailed knowledge of the oligosaccharides of recombinantly produced human glycoprotein therapeutics is a prerequisite for their use in patients. From the regulatory point of view (EMEA, FDA), the carbohydrates of therapeutic glycoproteins (e.g. EPO, IFN-β, therapeutic antibodies) play a key role in efficacy and safety (e.g. immunogenicity).
On cellular surfaces, glycans are typically conjugated to proteins (glycoproteins) and ceramides (glycosphingolipids) which are incorporated into the lipid bilayer. Glycoproteins are also in cellular secretions. Despite progress in high performance liquid chromatography (HPLC), lectin affinity chromatography, mass spectrometry (MS), and glycan microarrays, chemically defining a glycome, the complete list of glycan structures that occur in a cell, tissue, or organism, has been elusive. Studying glycosphingolipids (GSLs) has been especially challenging. In previous approaches to overcome the poor detection sensitivity during HPLC separation of GSLs, the component glycans were released from lipids and analyzed as fluorescent glycans by HPLC. See Wing et al., Anal Biochem, 2001, 298, 207-17 and Ohara et al., J Chromatogr, 1991, 586, 35-41. However, these methods provided no capability to explore GSLs recognition by glycan-binding proteins (GBPs). Thus, there is a need to identify improved methods for detecting and analyzing glycosphingolipids.
There have been a number of methods developed for glycan conjugation. See e.g., Luyai et al., Bioconjugate Chem, 2009, 20 (8), 1618-1624; Song et al., Chem Biol, 2009, 4(9), 741-750; and Song et al., Chem Biol, 2009, 16, 36-47.